This invention relates to antiparasitic agents and in particular to compounds related to the avermectins and milbemycins but having a novel substituent group at the 25-position and to a process for their preparation.
The avermectins are a group of broad spectrum antiparasitic agents referred to previously as the C-076 compounds. They are produced by fermenting a strain of the microorganism Streptomyces avermitilis ATCC 31267, 31271 or 31272 under aerobic conditions in an aqueous nutrient medium containing inorganic salts and assimilable sources of carbon and nitrogen. The morphological and cultural properties of the strains ATCC 31267, 31271 and 31272 are described in detail in British Patent Specification No. 1573955 which also describes the isolation and the chemical structure of the eight individual components which make up the C-076 complex. The milbemycins are structurally related macrolide antibiotics lacking the sugar residues at the 13-position. They are produced by fermentation, for example as described in British Patent Specification No. 1390336 and European Patent Application publication No. 0170006.
In our European Patent Application publication No. 0214731, published Mar. 18, 1987, the counterpart of U.S. application Ser. No. 886,867, filed Jul. 16, 1986, we disclose that by adding certain specified carboxylic acids, or derivatives thereof, to the fermentation of an avermectin producing organism it is possible to obtain novel compounds, related to the avermectins but having an unnatural substituent group at the 25-position in place of the isopropyl or sec-butyl group which is normally present.
The novel compounds produced are characterised in that the substituent group at the 25-position is alpha-branched i.e. the carbon atom attached to the C-25 ring position is a secondary carbon atom linked to two further carbon atoms.
In our co-pending U.S. patent application Ser. No. 107,825, filed Oct. 13, 1987, we describe and claim new mutant strains of the microorganism Streptomyces avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity. Said strains have been deposited in the American Type Culture Collection, Rockville, Md. under the designations Streptomyces avermitilis ATCC 53567 and ATCC 53568.
We have now discovered that, by using these new mutant strains of Streptomyces avermitilis it is possible to obtain a further range of novel avermectin derivatives, not previously obtainable, wherein the C-25 substituent is linked by an unbranched (primary) carbon atom. The novel compounds are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides. The compounds can be subjected to conventional chemical transformation reactions to obtain further novel semi-synthetic derivatives. Thus, according to the present invention there are provided compounds having the formula (I): 
wherein
X represents a single or a double bond; R1 is H or OH; provided that when X is a single bond, R1 is H or OH, and when X is a double bond, R1 is absent;
R2 is H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, alkoxy-alkyl or alkylthioalkyl containing from 1 to 6 carbon atoms in each alkyl or alkoxy group, wherein any of said alkyl, alkoxy alkenyl or alkynyl groups may be substituted by one or more halo atoms; or a C3-C8 cycloalkyl or C5-C8cycloalkenyl group, either of which may be substituted by methylene or one or more C1-C4 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may be substituted by one or more C1-C4 alkyl groups or halo atoms; or a group of the formula SR5 wherein R5 is C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C5-C8 cycloalkenyl, phenyl or substituted phenyl wherein the substituent is C1-C4 alkyl, C1-C4 alkoxy or halo, or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may be substituted by one or more C1-C4 alkyl groups or halo atoms;
R3 is hydrogen or methyl;
and R4 is H or a 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy group of the formula 
with the proviso that when R4 and R1 are both H and the double bond is absent, R2 is not H or CH3.
In the above definition, alkyl groups containing 3 or more carbon atoms may be straight or branched chain. Halo means fluoro, chloro, bromo or iodo.
The C-076 complex comprises eight distinct but closely related compounds described as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. The xe2x80x9caxe2x80x9d series of compounds refers to the natural avermectin wherein the 25-substituent is (S)-sec-butyl and the xe2x80x9cbxe2x80x9d series to those wherein the 25-substituent is isopropyl. The designations xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d refer to avermectins wherein the 5-substituent is methoxy or hydroxy, respectively, and the numeral xe2x80x9c1xe2x80x9d refers to avermectins wherein a double bond is present at the 22-23 position, and numeral xe2x80x9c2xe2x80x9d to avermectins having a hydrogen at the 22-position and hydroxy at the 23 position.
In this application, the xe2x80x9caxe2x80x9d and xe2x80x9cbxe2x80x9d identifiers have been dropped. Identifiers A1, A2, B1 and B2 have been retained to refer to non-natural avermectins having the structural features corresponding to those of the natural avermectins as noted above.
Preferred compounds of the formula (I) are those wherein R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy.
Also preferred are compounds of the formula (I) wherein R2 is SR5 and R5 is methyl or ethyl.
In another group of preferred compounds R2 is methyl, isopropyl or sec-butyl.
In a further group of preferred compounds R2 is branched C3-C8 alkyl group substituted by one or more halo atoms, particularly 1-(trifluorome thyl) ethyl.
In accordance with the invention the compounds of formula (I) wherein R1 is OH and the double bond is absent or wherein the double bond is present and R1 is absent and R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy are prepared by fermenting a Streptomyces avermitilis mutant organism ATCC 53567 or 53568, as described in U.S. patent application Ser. No. 107,825, in the presence of the appropriate carboxylic acid of the formula R2CH2CO2H, wherein R2 is as previously defined, or a salt, ester, or amide thereof or oxidative precursor therefor. The acid is added to the fermentation either at the time of inoculation or at intervals during the fermentation. Production of the compounds of formula (I) may be monitored by removing samples from the fermentation, extracting with an organic solvent and following the appearance of the compound of formula (I) by chromatography, for example using high pressure liquid chromatography. Incubation is continued until the yield of the compound of formula (I) has been maximised, generally for a period of from 12 to 16 days.
A preferred level of each addition of the carboxylic acid or derivative thereof is between 0.05 and 4.0 grams per litre. The best yields of the compounds of formula (I) are obtained by gradually adding the acid to the fermentation, for example by daily additions of the acid or derivative thereof over a period of several days. The acid may be added as a salt, such as the sodium or ammonium salt, or as an ester, such as the methyl or ethyl ester or as an amide, but is preferably added as the free acid. Alternative substrates which may be used in the fermentation are derivatives which are oxidative precursors for the carboxylic acids; thus, for example suitable substrates would be alcohols of the formula R2 (CH2)nOH or amine derivatives of the formula R2 (CH2)nNH2, wherein n is 2, 4 or 6, substituted lower alkanoic acids of the formula R2(CH2)nCO2H wherein n is 3 or 5 or aldehydes of the formula R2 (CH2)nCHO wherein n is 1, 3 or 5 and R2 is as previously defined. The media used for the fermentation may be a conventional complex media containing assimilable sources of carbon, nitrogen and other trace elements.
After fermentation for a period of several days at a temperature preferably in the range of from 24 to 33xc2x0 C., the fermentation broth is centrifuged or filtered and the mycelial cake is extracted with acetone or methanol. The solvent extract is concentrated and the desired product is then extracted into a water-immiscible organic solvent, such as methylene chloride, ethyl acetate, chloroform, butanol or methyl isobutyl ketone. The solvent extract is concentrated and the crude product containing the compounds of formula (I) is further purified as necessary by chromatography, for example using preparative reverse phase, high pressure liquid chromatography.
The product is generally obtained as a mixture of the compounds of formula (I) wherein R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy, R1 is OH and the double bond absent or R1 is absent and the double bond is present and wherein R3 is H or CH3; however the proportions can vary depending on the particular carboxylic acid employed and the conditions used in the fermentation.
We have found that a range of carboxylic acids as defined by R2 CH2CO2H may be added to the fermentation to yield avermectins having a novel substituent group at the 25-position. Examples of particular acids which may be employed include the following:
methylthioacetic acid
ethylthioacetic acid
3-methylbutyric acid
3-trifluoromethyl butyric acid
3-methylpentanoic acid
n-butyric acid
cyclopentane acetic acid
thiophene-3-acetic acid
and propionic acid.
In one particular and preferred aspect of the invention, the fermentation is performed in the presence of methylthioacetic acid to yield predominantly the compound of formula (I) wherein R1 is OH, the double bond is absent, R2 is SCH3, R3 is CH3 and R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to herein as 25-methylthiomethyl avermectin A2.
In another preferred aspect of the invention, the fermentation is performed in the presence of propionic acid to yield predominantly the compound of formula (I) wherein R1 is OH, the double bond is absent, R2 is CH3, R3 is CH3 and R4 is 4xe2x80x2(-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to herein as 25-ethyl avermectin A2.
In a further preferred aspect of the invention the fermentation is performed in the presence of 3-methylbutyric acid to yield predominantly the compound of formula (I) wherein R1 is absent, the double bond is present, R2 is isopropyl, R3 is H and R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to herein as 25-isobutyl avermectin B1.
In a further preferred aspect of the invention, the fermentation is performed in the presence of 3-trifluoromethyl butyric acid to yield predominantly the compounds of formula (I) wherein R1 is OH, the double bond is absent, R2 is 1-(trifluoro-methyl)ethyl, R4 is 4xe2x80x2-(alpha-L-oleandrosyl)alpha-4-oleandrosyloxy and R3 is CH3 or H referred to herein as 25-(2-trifluoromethyl-propyl)avermectin A2 and B2 respectively.
Compounds of the formula (I) wherein the double bond is present and R1 is absent may alternatively be prepared from the corresponding compound of formula (I) wherein R1 is OH and the double bond is absent by a dehydration reaction. The reaction is performed by first selectively protecting the hydroxyl groups at the 5 and 4xe2x80x3 positions, e.g. as the t-butyldimethylsilyloxy acetyl derivative, then reacting with a substituted thiocarbonyl halide, such as (4-methylphenoxy)thiocarbonyl chloride, followed by heating in a high boiling point solvent, e.g. trichlorobenzene, to effect the dehydration. The product is finally deprotected to give the unsaturated compound. These steps together with appropriate reagents and reaction conditions are described in U.S. Pat. No. 4,328,335.
The compounds of formula I wherein R3 is H may also be prepared from the corresponding compounds wherein R3 is CH3 by demethylation. This reaction is achieved by treating the 5-methoxy compound, or a suitably protected derivative thereof, with mercuric acetate and hydrolysing the resulting 3-acetoxy enol ether with dilute acid to give the 5-keto compound. This is then reduced using, for example, sodium borohydride to yield the 5-hyaroxy derivative. Appropriate reagents and reaction conditions for these steps are described in U.S. Pat. No. 4,423,209.
The compounds of-formula I wherein R1 is H and the double bond is absent can be prepared from the corresponding compound wherein the double bond is present and R1 is absent, by selective catalytic hydrogenation using an appropriate catalyst. For example the reduction may be achieved using tris(triphenylphosphine)rhodium (I) chloride as described in European patent application publication No. 0001689.
The compounds of formula (I) wherein R4 is H are prepared from the corresponding compounds wherein R4 is 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy by removing the 4xe2x80x2-(alpha-L-oleandrosyl)-alpha-L-oleandrose group by mild hydrolysis with an acid in an aqueous organic solvent to yield the aglycone having a hydroxy group at the 13-position; this is then halogenated, for example by reaction with a benzene esulphonyl halide, to yield the 13-deoxy-13-halo derivative which is finally selectively reduced, for example using tributyltin hydride. In order to avoid unwanted side reactions it is desirable to protect any other hydroxy groups which may be present, for example using a tert-butyldimethylsilyl group. This is then readily removed after the halogenation or reduction step by treatment with methanol containing a trace of acid. All these steps together with appropriate reagents and reaction conditions for their performance are described in European patent application publication no. 0002615.
The compounds of the invention are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.
Thus the compounds are effective in treating a variety of conditions caused by endoparasites including, in particular, helminthiasis which is most frequently caused by a group of parasitic worms described as nematodes and which can cause severe economic losses in swine, sheep, horses and cattle as well as affecting domestic animals and poultry. The compounds are also effective against other nematodes which affect various species of animals including, for example, Dirofilaria in dogs and various parasites which can infect humans including gastrointestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius and parasites which are found in the blood or other tissues and organs such as filiarial worms and the extra intestinal stages of Strongyloides and Trichinella.
The compounds are also of value in treating, including preventing, ectoparasite infections including in particular arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating dipterous larvae which can affect cattle and horses.
The compounds are also insecticides active against household pests such as the cockroach, clothes moth, carpet beetle and the housefly as well as being useful against insect pests of stored grain and of agricultural plants such as spider mites, aphids, caterpillars and against migratory orthopterans such as locusts.
The compounds of formula (I) are administered as a formulation appropriate to the specific use envisaged and to the particular species of host animal being treated and the parasite or insect involved. For use as an anthelmintic the compounds may be administered orally in the form of a capsule, bolus, tablet or preferably a liquid drench, or alternatively, they may be administered by injection or as an implant. Such formulations are prepared in a conventional manner in accordance with standard veterinary practice. Thus, capsules, boluses or tablets may be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier, additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, magnesium stearate etc. A drench formulation may be prepared by dispersing the active ingredient in an aqueous solution together with dispersing or wetting agents etc. and injectable formulations may be prepared in the form of a sterile solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. These formulations will vary with regard to the weight of active compound depending on the species of host animal to be treated, the severity and type of infection and the body weight of the host. Generally for oral administration a dose of from about 0.001 to 10 mg per Kg of animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory but of course there can be instances where higher or lower dosage ranges are indicated and such are within the scope of this invention.
As an alternative the compounds may be administered with the animal feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.
For use as an insecticide and for treating agricultural pests the compounds are applied as sprays, dusts, emulsions and the like in accordance with standard agricultural practice.
For human use the compounds are administered as a pharmaceutically acceptable formulation in accordance with normal medical practice.
The invention is illustrated by the following Examples in which Examples 1 to 8 are Examples of the preparation of compounds of the formula (I), Example 9 is an example of a drench formulation and Examples 10 and 11 illustrate the antiparasitic and insecticidal activity of the compounds.